Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Effect of pseudobactin 358 production by Pseudomonas putida WCS358 on suppression of fusarium wilt of carnations by nonpathogenic Fusarium oxysporum Fo47.

Nonpathogenic Fusarium oxysporum Fo47b10 combined with Pseudomonas putida WCS358 efficiently suppressed fusarium wilt of carnations grown in soilless culture. This suppression was significantly higher than that obtained by inoculation of either antagonistic microorganism alone. The increased suppression obtained by Fo47b10 combined with WCS358 only occurred when Fo47b10 was introduced at a density high enough (at least 10 times higher than that of the pathogen) to be efficient on its own. P. putida WCS358 had no effect on disease severity when inoculated on its own but significantly improved the control achieved with nonpathogenic F. oxysporum Fo47b10. In contrast, a siderophore-negative mutant of WCS358 had no effect on disease severity even in the presence of Fo47b10. Since the densities of both bacterial strains at the root level were similar, the difference between the wild-type WCS358 and the siderophore-negative mutant with regard to the control of fusarium wilt was related to the production of pseudobactin 358. The production of pseudobactin 358 appeared to be responsible for the increased suppression by Fo47b10 combined with WCS358 relative to that with Fo47b10 alone.     

Ability of Nonpathogenic Fusarium oxysporum Strain Fo47 To Induce Resistance against Pythium ultimum Infection in Cucumber

The influence exerted by nonpathogenic Fusarium oxysporum strain Fo47 in triggering cucumber protection against infection by Pythium ultimum was investigated ultrastructurally. Macroscopic and microscopic observations of the pathogen colony in dual cultures revealed that reduction of Pythium growth was associated with marked disorders, including generalized disorganization of the host cytoplasm, retraction of the plasmalemma, and complete loss of the protoplasm. Cytochemical labeling of cellulose with an exoglucanase-gold complex showed that the cellulose component of the host cell walls was structurally preserved at a time when the host cytoplasm had undergone complete disorganization. A similar antagonistic process was observed at the root cell surface. Most striking and interesting was the finding that mycoparasitism, as evidenced by the frequent occurrence of Fo47 hyphae within nearly empty cells of the pathogen, occurred not only at the root surface but also within the invaded root tissues. The specific labeling pattern obtained with the exoglucanase-gold complex confirmed that Fo47 successfully penetrated cells of the pathogen, both in the rhizosphere and inside the root tissues. Pythium cells that could evade the first defensive line in the rhizosphere could penetrate the root epidermis, but their growth was restricted to the outermost tissues. Positive correlations between Fo47 treatment and induced resistance to infection by P. ultimum in cucumber were confirmed by (i) the reduction of pathogen viability; (ii) the elaboration of newly formed barriers, a phenomenon which was not seen in Fo47-free plants, where the pathogen proliferated in all root tissues within a few days; and (iii) the occlusion of intercellular spaces with a dense material likely enriched in phenolics. Taken together, our observations provide the first convincing evidence that Fo47 exerts a direct inhibitory effect on P. ultimum through a combination of antibiosis and mycoparasitism, in addition to being a strong inducer of plant defense reactions.     

Extracellular Chitinases of Fluorescent Pseudomonads Antifungal to Fusarium oxysporum f. sp. dianthi Causing Carnation Wilt

Vascular wilt of carnation caused by Fusarium oxysporum f. sp. dianthi (Prill. & Delacr.) W. C. Synder & H.N. Hans inflicts substantial yield and quality loss to the crop. Mycolytic enzymes such as chitinases are antifungal and contribute significantly to the antagonistic activity of fluorescent pseudomonads belonging to plant-growth-promoting rhizobacteria. Fluorescent pseudomonads antagonistic to the vascular wilt pathogen were studied for their ability to grow and produce chitinases on different substrates. Bacterial cells grown on chitin-containing media showed enhanced growth and enzyme production with increased anti-fungal activity against the pathogen. Furthermore, the cell-free bacterial culture filtrate from chitin-containing media also significantly inhibited the mycelial growth. Both the strains and their cell-free culture filtrate from chitin-amended media showed the formation of lytic zones on chitin agar, indicating chitinolytic ability. Extracellular proteins of highly antagonistic bacterial strain were isolated from cell-free extracts of media amended with chitin and fungal cell wall. These cell-free conditioned media contained one to seven polypeptides. Western blot analysis revealed two isoforms of chitinase with molecular masses of 43 and 18.5 kDa. Further plate assay for mycelial growth inhibition showed the 43-kDa protein to be antifungal. The foregoing studies clearly established the significance of chitinases in the antagonism of fluorescent pseudomonads, showing avenues for possible exploitation in carnation wilt management.     

Fungitoxic phenols from carnation (Dianthus caryophyllus) effective against Fusarium oxysporum f. sp. dianthi

The phenol compositions of two cultivars of carnation (Dianthus caryophyllus) namely "Gloriana" and "Roland", which are partially and highly resistant, respectively, to Fusarium oxysporum f. sp. dianthi have been investigated with the aim of determining if endogenous phenols could have an anti-fungal effect against the pathogen. Analyses were performed on healthy and F. oxysporum-inoculated in vitro tissues, and on in vivo plants. Two benzoic acid derivatives, protocatechuic acid (3,4-dihydroxybenzoic acid) and vanillic acid (4-hydroxy-3-methoxybenzoic acid), were found within healthy and inoculated tissues of both cultivars, together with the flavonol glycoside peltatoside (3-[6-O-(alpha-L-arabinopyranosyl)-beta-D-glucopyranosyl] quercetin). These molecules proved to be only slightly inhibitory towards the pathogen. 2,6-Dimethoxybenzoic acid was detected in small amounts only in the inoculated cultivar "Gloriana", while the highly resistant cultivar "Roland" showed the presence of the flavone datiscetin (3,5,7,2'-tetrahydroxyflavone). The latter compound exhibited an appreciable fungitoxic activity towards F. oxysporum f. sp. dianthi.     

香蕉枯萎病拮抗放线菌Da08006的筛选与鉴定

以尖孢镰刀菌4号小种为指示菌株,对海南尖峰岭原始森林和发病香蕉园健康植株根际土壤的放线菌进行筛选,获得一株具有较强拮抗作用、遗传稳定的放线菌Da08006.通过形态特征、生理生化特征、16S rDNA序列及其系统发育分析研究,鉴定菌株Da08006为Streptomyces morookaense.     

Production of Intraspecific Hybrids of Fusarium oxysporum f. sp. radicis-lycoperisici and Fusarium oxysporum f. sp. lycopersici by Protoplast Fusions

The fusion of protoplasts from the cycloheximide-resistant mutant FOL(C) of Fusarium oxysporum f. sp. lycopersici (FOL) and the mycostatin-resistant mutant FORL(M) of F. oxysporum f. sp. radicis-lycopersici (FORL), produced hybrids which expressed significant differences from the parents in their pathogenicity and growth and in the electrophoretic separation patterns of their proteins, enzymes and isoenzymes. The results suggest a transformed genetic basis for these altered expressions and the feasibility of using protoplast fusion technology for examining the biology of pathogenicity genes and for elucidating the disease and virulence potential for new races from within hybridisable taxa of Fusarium spp. Such information would be useful for the design and development of long-term control systems for Fusarium diseases, particularly in breeding programs for disease resistance in crops.     

Antagonistic effects of soil bacteria onFusarium oxysporum Schlecht f. sp.dianthi (Prill and Del.) Snyd. and Hans

Summary Fusarium oxysporum f. sp.dianthi, pathogenic on carnation plants is very sensitive toBacillus subtilis M51 inhibition.Fusarium oxysporum disease (fusariosis) is prevented for a period of two months after treatment of plants withBacillus subtilis M51. The persistence ofB. subtilis M51, marked for selenomycin resistance (MZ51) and inoculated on the roots of carnation cuttings was studied. Soil used was two types: naturally infested withFusarium oxysporum and free from this pathogen. Bacterial cells presence on the roots was detected by direct plating and the presence of the pathogen in the roots was investigated by histological assays. Evidence gathered by these procedures suggest that plant protection is dependent on the physical presence ofB. subtilis M51 cells on the roots.     

The Effect of Temperature on Symptom Expression and Colonization in Resistant and Susceptible Carnation Cultivars Infected with Fusarium oxysporum f. sp. dianthi

Six commercial carnation cultivars were inoculated with Fusarium oxysporum f. sp. dianthi race 2, and grown under three different temperature regimes. Colonization by the pathogen and development of wilt symptoms were assessed at intervals up to 40 days. No symptoms and very little colonization were seen in any of the cultivars at 14–15°C. At a temperature of 22°C, the cultivars were clearly differentiated into three groups: those with resistance, partial resistance or susceptibility to the pathogen depending on the severity of symptoms and the extent of fungal colonization. Symptom severity was associated with the extent of colonization. This differentiation was not seen at 26°C, when all cultivars except the most resistant, cv.‘Carrier 929′, rapidly became diseased and died by 23 days after inoculation. ‘Carrier 929’ also showed some wilt symptoms at this temperature and was colonized throughout the height of the stem after 40 days. The pathogen caused disease at 26°C by a combination of vascular wilting and stem base and root rotting. Fungal colonization was assayed at 22°C by the dilution plate/homogenization method and by estimation of fungal chitin in a highly resistant (‘Carrier 929′) and in a highly susceptible (‘Red Baron’) cultivar. Both methods of assay gave similar results. In ‘Red Baron’, colonization increased slowly up to 20 days after inoculation then progressed rapidly, closely following the development of severe wilt symptoms. In ‘Carrier 929’, colonization remained very low. The low level of fungal biomass in ‘Carrier 929’ compared with ‘Red Baron’ indicated that the former cultivar showed true resistance as opposed to tolerance to the disease.     

Mineral Uptake in Fusarium oxysporum f. sp. vasinfectum

A method for growing Fusarium oxysporum, a mycelial fungus, and a technique for its use in mineral uptake studies have been described. Some general characteristics of the uptake process were determined. The fungus, grown for 54 hours, was found to take up as much K as 15 to 20 meq/100 g dry weight in 2 to 4 hours from a solution of 5 meq/l KCl. Approximately 3 to 5 meq of this uptake was readily removed by a CaCl₂ rinse. The uptake was only slightly sensitive to pH over the range of 4 to 9. Below pH 4 uptake dropped rapidly. The age of the culture appeared to be the dominant factor in determining the rate of uptake. In contrast to other fungi, the presence of glucose during uptake was detrimental to K uptake. Conditions unfavorable for metabolic activity as low temperature, anaerobiosis, or the presence of DNP markedly reduced the uptake rate. Although the fungus took up Na from single salt solutions nearly as well as K, the latter ion was much preferred in mixtures of the two ions. The organism showed no significant metabolic uptake of Ca or Cl. During uptake from KCl solutions, the organic acid content increased. The increase, chiefly in succinic acid and to a lesser extent in acetic and citric acids, amounted to about half the K uptake. The remainder of the K taken up was correlated with a roughly equivalent efflux of cellular Mg.     

Dispensable chromosomes in Fusarium oxysporum f. sp. lycopersici

The genomes of many filamentous fungi consist of a ‘core’ part containing conserved genes essential for normal development as well as conditionally dispensable (CD) or lineage‐specific (LS) chromosomes. In the plant‐pathogenic fungus Fusarium oxysporum f. sp. lycopersici, one LS chromosome harbours effector genes that contribute to pathogenicity. We employed flow cytometry to select for events of spontaneous (partial) loss of either the two smallest LS chromosomes or two different core chromosomes. We determined the rate of spontaneous loss of the ‘effector’ LS chromosome in vitro at around 1 in 35 000 spores. In addition, a viable strain was obtained lacking chromosome 12, which is considered to be a part of the core genome. We also isolated strains carrying approximately 1‐Mb deletions in the LS chromosomes and in the dispensable core chromosome. The large core chromosome 1 was never observed to sustain deletions over 200 kb. Whole‐genome sequencing revealed that some of the sites at which the deletions occurred were the same in several independent strains obtained for the two chromosomes tested, indicating the existence of deletion hotspots. For the core chromosome, this deletion hotspot was the site of insertion of the marker used to select for loss events. Loss of the core chromosome did not affect pathogenicity, whereas loss of the effector chromosome led to a complete loss of pathogenicity.     

Race Differentiation in Fusarium oxysporum f.sp. chrysanthemi

The pathogenicity of different isolates of Fusarium oxysporum obtained from plants of Gerbera (Gerbera jamesonii), Chrysanthemum (Chrysanthemum morifolium), Paris daisy (Argyranthemum frutescens) and African daisy (Osteospermum sp.), all in the family Asteraceae, was tested on different cultivars of these hosts, to assess their pathogenicity. The reactions were compared with those of isolates of F. oxysporum f. sp. chrysanthemi and of f.sp. tracheiphilum obtained from the American Type Culture Collection. We found that isolates of F. oxysporum f. sp. chrysanthemi can be distinguished as three physiological races on the basis of their pathogenicity to the panel of differential cultivars. Sequencing of the intergenic spacer (IGS) region of ribosomal DNA (rDNA) and phylogenetic analysis showed that the Fusarium races fell into three phylogenetic groups, which coincided with those observed in pathogenicity tests. Analysis of the IGS sequences revealed a high degree of similarity among strains from Italy and Spain from different host species, suggesting that recent outbreaks in these ornamentals were probably caused by introduction of infected nursery material from a common origin.     

Effect of flavonoids on the development of Fusarium oxysporum f. sp. lycopersici

Abstract

In the present study the effect of flavonoid compounds on the germination and fungal growth of the soil-borne tomato pathogen Fusarium oxysporum f. sp. lycopersici was studied. Out of 12 flavonoid compounds only myricetin and luteolin exhibited a low stimulating activity on microconidia germination of Fusarium oxysporum f. sp. lycopersici, whereas the other flavonoids tested were inactive when applied at five different concentrations. In our study the tested flavonoids affect fungal growth differently to microconidia germination. Individual flavonoid concentrations resulted in a small increase of fungal growth, but the lowest flavonoid concentrations showed an inhibiting effect on fungal growth for all flavonoids tested. There is evidence to suggest, that low flavonoid concentrations exhibit slight antimicrobial properties against Fusarium oxysporum f. sp. lycopersici.     

Selection of Chickpea-Rhizosphere-Competent Pseudomonas fluorescens NBRI1303 Antagonistic to Fusarium oxysporum f. sp. ciceri,Rhizoctonia bataticola and Pythium sp.

A procedure that consumes less screening time was developed for screening chickpea rhizosphere-competent bacteria for suppression of the chickpea pathogenic fungi Fusarium oxysporum f. sp. ciceri, Rhizoctonia bataticola and Pythium sp. Of the 478 bacteria obtained by random selection of the predominant, morphologically distinct colonies, 386 strains that effectively colonize chickpea roots could be divided broadly into three different groups. The first group consisted of 44 good chickpea rhizosphere colonizers with 10⁷ to 10⁸ colony-forming units (CFU)/g root; the second group consisted of 253 medium chickpea rhizosphere colonizers with 10⁴ to 10⁶ CFU/g root; and the third group consisted of 89 poor chickpea rhizosphere colonizers with 10⁰ (nondetectable) to 10³ CFU/g root. Forty-four Rif^r strains from the first group of good chickpea rhizosphere colonizers were further screened for their in vitro biocontrol activity against F. oxysporum f. sp. ciceri, R. bataticola, and Pythium sp. One bacterial strain was selected for further work because of its unique ability to inhibit all three fungi and its good chickpea rhizosphere colonization ability. This is the first report of a single biocontrol bacterium active against three most devastating pathogenic fungi of chickpea. In a greenhouse test, chickpea seed bacterization with P. fluorescens NBRI1303 increased the germination of seedlings by 25%, reduced the number of diseased plants by 45%, compared with nonbacterized controls. Increases in seedling dry weight, shoot length, and root length ranged from 16% to 18%. Significant growth increases in shoot length, dry weight, and grain yield, averaging 11.59%, 17.58%, and 22.61% respectively above untreated controls, were attained in field trials in Agra and Jhansi. A rifampicin-resistant mutant P. fluorescens NBRI1303R of the P. fluorescens NBRI1303, used to monitor chickpea root colonization, confirmed the rapid and aggressive colonization by the bacterium, making it a potential biocontrol agent against chickpea phytopathogenic fungi. The results, demonstrating an increase in the efficiency of screening and detection of plant beneficial strains, should greatly benefit future studies. Received: 23 December 1996 / Accepted: 28 January 1997     

Kaempferide triglycoside: a possible factor of resistance of carnation (Dianthus caryophyllus) to Fusarium oxysporum f. sp. dianthi

A kaempferide triglycoside has been found as a constitutive component in an uninfected carnation (Dianthus caryophyllus) of the cultivar Novada. The chemical structure has been determined mainly by the use of spectroscopic methods, including 2D NMR experiments. It showed a strong activity in restricting fungal parasite development, which could contribute to the known ability of carnation cv. Novada to resist to Fusarium oxysporum f. sp. dianthi infection.     

西瓜枯萎病菌的快速检测与鉴定

通过西瓜枯萎病菌与其他专化型枯萎病菌及瓜类几种重要病原菌的比较基因组分析,获得了西瓜枯萎病菌的基因组特异序列。在此基础上,设计出特异引物,筛选可扩增出西瓜枯萎病菌特异性DNA条带的引物。将特异性引物和尖孢镰刀菌专化型的通用引物W106R/W106S结合,建立双重PCR检测体系。该双重PCR检测体系可以在一次PCR反应中快速、准确的检测出西瓜枯萎病菌,为通过分子方法快速鉴定西瓜枯萎病菌提供技术支持。     

Selection and differentiation of Bacillus spp. Antagonistic to Fusarium oxysporum f.sp. lycopersici and Alternaria solani infecting Tomato

Antagonistic Bacillus spp. displaying in vitro production of siderophore, chitinase, and β-1,3-glucanase were identified from dual culture assays. In independent greenhouse studies, seed bacterization and soil application of Bacillus atrophaeus S2BC-2 challenge inoculated with Fusarium oxysporum f.sp. lycopersici (FOL) and Alternaria solani (AS) recorded low percent disease index of 25.3 and 28.7, respectively, over nonbacterised pathogen control (44.3 and 56.4). The low disease incidence corroborated with tomato growth promotion with high vigor index (8,041.2) and fresh plant weight (82.5 g) on challenge inoculation with FOL. Analysis of root and leaf samples in rhizobacterial treatment challenged with FOL and AS revealed maximum induction of chitinase (1.9 and 1.7 U/mg of protein, respectively) and β-1,3-glucanase (23.5 and 19.2 U/mg of protein, respectively). In native gel activity assays, the rhizobacterial treatment on challenge inoculation strongly expressed three high intensity PO isoforms along with one low intensity isoform. In studies on genetic diversity of the Bacillus strains by repetitive extragenomic palindromic-polymerase chain reaction (REP-PCR) and amplified rDNA restriction analysis (ARDRA) patterns, ARDRA was more highly discriminant than REP-PCR and allowed grouping of the strains and differentiation of the antagonistic strains from other isolates.